ascorbic-acid and Melanoma

Vitamin C is implicated in various bodily functions due to its unique properties in redox homeostasis. Moreover, vitamin C also plays a great role in restoring the activity of 2-oxoglutarate and Fe

Topics: Ascorbic Acid; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Breast Neoplasms; Carcinogenesis; Dehydroascorbic Acid; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Epigenesis, Genetic; Female; Glioma; Glucose Transport Proteins, Facilitative; Hematologic Neoplasms; Homeostasis; Humans; Hypoxia-Inducible Factor 1; Ketoglutaric Acids; Male; Melanoma; Mixed Function Oxygenases; Neoplasms; Oxidation-Reduction; Polymorphism, Genetic; Prostatic Neoplasms; Proto-Oncogene Proteins; Sodium-Coupled Vitamin C Transporters; Vitamins

Intravenous application of high-dose ascorbate (vitamin C) has been used in complementary medicine since the 1970s to treat cancer patients. In recent years it became evident that high-dose ascorbate in the millimolar range bears selective cytotoxic effects on cancer cells in vitro and in vivo. This anticancer effect is dose dependent, catalyzed by serum components and mediated by reactive oxygen species and ascorbyl radicals, making ascorbate a pro-oxidative pro-drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. It further depends on HIF-1 signaling and oxygen pressure, and shows a strong epigenetic signature (alteration of DNA-methylation and induction of tumor-suppressing microRNAs in cancer cells). The detailed understanding of ascorbate-induced antiproliferative molecular mechanisms warrants in-depth preclinical evaluation in cancer-bearing animal models for the optimization of an efficacious therapy regimen (e.g., combination with hyperbaric oxygen or O2-sensitizers) that subsequently need to be evaluated in clinical trials.

Topics: Administration, Oral; Animals; Antioxidants; Ascorbic Acid; Cell Survival; Combined Modality Therapy; Complementary Therapies; Dose-Response Relationship, Drug; Drug Approval; Epigenesis, Genetic; European Union; Humans; Infusions, Intravenous; Melanoma; MicroRNAs; Phytotherapy; Reactive Oxygen Species; Risk Management; Treatment Outcome; Tumor Cells, Cultured

Melanoma is the most serious form of skin cancer affecting mostly people of Caucasian origin and is associated with high exposure to solar ultraviolet (UV) radiation. Antioxidants in the diet are thought to prevent UV-induced DNA damage and oxidative stress and laboratory-based studies have shown that high antioxidant intakes inhibit melanoma development. Corresponding epidemiological evidence is inconsistent, however. We therefore reviewed results from prospective observational studies and randomized controlled trials (RCTs) to clarify whether consumption of antioxidant vitamin C, E (tocopherol), and A (retinol), carotenoids and selenium, as food, supplements, or both, or high fruit and vegetable intake, reduce the incidence of cutaneous melanoma. A total of 9 studies (2 cohort, 1 nested case-control, 6 RCTs) were included. Neither antioxidant nutrients, individually or combined, nor fruit and vegetable intake showed any strong and significant associations with melanoma, though the number of relevant studies was limited and several had methodological shortcomings. In particular, melanoma was not a primary disease outcome in any of the RCTs and therefore, none adequately accounted for potential confounding by sun exposure. In conclusion, available evidence is currently inadequate to assess possible beneficial effects of antioxidant intake on melanoma risk.

Topics: Antioxidants; Ascorbic Acid; Carotenoids; Databases, Factual; Dietary Supplements; DNA Damage; Fruit; Humans; Melanoma; Melanoma, Cutaneous Malignant; Observational Studies as Topic; Oxidative Stress; Randomized Controlled Trials as Topic; Selenium; Skin Neoplasms; Vegetables; Vitamin A; Vitamin E

Experimental animal studies have unambiguously demonstrated that topical sunscreens can prevent squamous cell carcinoma and photoaging (damage of collagen and elastic fibers of the skin). Although data from clinical studies and surrogate markers also indicate such photoprotective effects in man, there is a lack of controlled, prospective clinical trials to provide definite evidence in man. Because of inadequate data, no definite conclusions can be drawn about the cancer-preventive activity of topical use of sunscreens against basal cell carcinoma and malignant melanoma.

Topics: Administration, Topical; Adult; Animals; Antioxidants; Ascorbic Acid; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Disease Models, Animal; Humans; Melanoma; Mice; Mice, Hairless; Neoplasms, Radiation-Induced; Prospective Studies; Randomized Controlled Trials as Topic; Skin Aging; Skin Neoplasms; Sunscreening Agents; Time Factors; Ultraviolet Rays; Vitamin E

Topics: Animals; Anticarcinogenic Agents; Antioxidants; Ascorbic Acid; beta Carotene; Carotenoids; Case-Control Studies; Cell Transformation, Neoplastic; Diet; Free Radicals; Guinea Pigs; Humans; Incidence; Lung Neoplasms; Melanoma; Mice; Mice, Hairless; Models, Chemical; Neoplasms; Neoplasms, Radiation-Induced; Oxygen; Partial Pressure; Prospective Studies; Reactive Oxygen Species; Retrospective Studies; Selenium; Singlet Oxygen; Skin Neoplasms; Smoking; Structure-Activity Relationship; Ultraviolet Rays; Vegetables; Vitamin E

Topics: Amino Acids; Animals; Ascorbic Acid; Cell Division; Cricetinae; Dietary Fats; Dietary Proteins; Humans; Melanoma; Mice; Nucleosides; Nutritional Physiological Phenomena; Pyridoxine; Skin Neoplasms; Trace Elements; Vitamin A; Vitamin D; Vitamin K

Nonspecific enhancers of resistance may include (1) viral interference, (2) interferon, (3) interferon inducers, (4) bacterial interference, (5) bacterial products such as Coley's "toxins," endotoxins, or staphylococcal, BCG, and Corynebacterium parvum vaccines, (6) transfer factor, and (7) well-defined chemicals such as dinitrochlorbenzene, levamisole, and vitamin C. These are discussed only as they have been applied to man to learn whether or not they have enhanced his ability to resist infections and growth of tumors. Preliminary studies suggest that a variety of relatively safe and effective nonspecific enhancers may soon be available for clinical use.

Topics: Adjuvants, Immunologic; Animals; Ascorbic Acid; Bacteria; BCG Vaccine; Dinitrochlorobenzene; Endotoxins; Humans; Immunity; Immunity, Cellular; Interferon Inducers; Interferons; Levamisole; Melanoma; Mice; Propionibacterium acnes; Rats; Respiratory Tract Infections; Skin Neoplasms; Staphylococcal Vaccines; Transfer Factor; Viral Interference

We conducted a single institution phase II trial to evaluate the tolerability and effectiveness of therapy with arsenic trioxide (ATO) and ascorbic acid (AA) with temozolomide (TMZ) in patients with advanced melanoma. Planned enrollment was for 40 patients. Eligible patients were required to have metastatic melanoma with or without brain metastases, an ECOG performance status of 0-2, and adequate organ function. The primary endpoints were overall response rate and degree of grade 4 toxicity. ATO was administered as a loading dose at 0.25 mg/kg/day for 5 days during week 0, and then twice weekly at 0.35 mg/kg during an 8-week cycle. Each infusion of ATO was followed by an infusion of 1000 mg of AA. TMZ was given at the standard dose of 200 mg/m for 5 days during weeks 1 and 5 of each cycle. Eleven patients were enrolled with 10 evaluable for response, five with central nervous system disease. No responses were seen among the first 10 patients and on the basis of a predetermined stopping rule, the trial was terminated. Common grade 1 and 2 side effects included nausea and vomiting (10), fatigue (six), edema (six), rash (six), and elevated AST or ALT (six). Grade 3 and 4 side effects included nausea and vomiting (three), elevated AST or ALT (two), seizure (one), and renal failure (one). This is the first trial combining ATO with chemotherapy in a solid tumor. ATO and AA were administered in the outpatient setting with TMZ in 11 patients with an acceptable side effect profile. No responses were seen in the first 10 evaluable patients leading to early closure of the study. Further studies using ATO and AA with TMZ with this dosing schedule in advanced melanoma are not warranted.

Topics: Adult; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Arsenic Trioxide; Arsenicals; Ascorbic Acid; Brain Neoplasms; Dacarbazine; Female; Humans; Male; Melanoma; Middle Aged; Oxides; Skin Neoplasms; Temozolomide

Zilascorb(2H) is a benzaldehyde derivative giving rise to strong protein synthesis inhibition. It has shown antitumor activity against human malignant melanoma grown as xenografts in nude mice. The effect was manifest only after prolonged daily treatment and was quickly reversible when treatment was stopped. Drug-induced fever was the dose-limiting toxicity observed during clinical phase I studies of zilascorb(2H). The object of the present study was to assess antitumor activity, safety and tolerability of the drug in melanoma patients. Sixteen patients with disseminated malignant melanoma were included, all presenting with WHO performance status 0-2 and adequate organ functions. Previous chemo- or radiotherapy was accepted, while patients with known CNS metastases were excluded. Due to its low solubility and quickly reversible activity, zilascorb(2H) 1400 mg was infused by the patients twice daily through a venous access port for up to 12 weeks. Induction of tumor regression was demonstrated in one patient, who was, however, withdrawn from treatment after 2 weeks because of recurrent fever and fatigue. All the 12 patients evaluable for antitumor activity had progressive disease. Zilascorb(2H) was well tolerated, except for fever reactions and reversible liver toxicity. Most patients learned quickly how to handle a venous access port, but daily self-administration of i.v. infusions became too cumbersome to justify further patient inclusion despite the tumor regression observed. We conclude that zilascorb(2H) seems to have the potential for antitumor activity in metastatic malignant melanoma and is well tolerated. Daily self-administration of drug infusions is not desirable for long periods and zilascorb(2H) tablets have been developed. Because of its favorable toxicity profile, especially compared to other protein synthesis inhibitors, zilascorb(2H) may be particularly interesting for combinations with other anticancer drugs.

Topics: Adult; Aged; Antineoplastic Agents; Ascorbic Acid; Benzylidene Compounds; Drug Administration Schedule; Female; Humans; Male; Melanoma; Middle Aged; Protein Synthesis Inhibitors

Oxidative stress is a barrier of migration and metastasis for malignant melanoma cells. Consequently, reducing oxidative stress with the antioxidant N-acetylcysteine (NAC) stimulates melanoma cell migration in vitro and metastasis in vivo. However, it is not yet known whether the NAC effect is shared with other antioxidants. Here, we screened 104 redox-active compounds and identify 27 that increase migration of human malignant melanoma cells in two doses. Validation experiments in four cell lines and four drug doses resulted in a list of 18 compounds which were ranked based on their ability to increase migration and reduce ROS levels; vitamin C (VitC) ranked as number one, followed by the vitamin E analogue Trolox and several carotenoids and Vitamin A-related compounds. Four diet-relevant compounds from this list-VitC, β-carotene, retinyl palmitate, and canthaxanthin-were selected and found to accelerate metastasis in mice with BRAF

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Humans; Melanoma; Melanoma, Cutaneous Malignant; Mice; Reactive Oxygen Species; Vitamin A; Vitamins

Vitamin C (VC)-based cancer therapy is a promising therapeutic approach for a variety of cancers due to its profound effects on redox reactions and metabolic pathways. However, high administration dosage of VC for necessary therapeutic efficacy for cancers increases the risk of overt side effects and limits its clinical use. Here, we show cutaneous blue light irradiation can specifically upregulate the sodium-dependent vitamin C transporter 2 (SVCT2) of the tumor and increase effectively the VC concentration at the tumor sites by an overall low dosage administration. In the mouse melanoma model, blue light stimulates the SVCT2 expression through the nuclear factor-kappa B (NF-κB) signaling pathway both in vitro and in vivo. The increased cellular VC together with Fe

Topics: Animals; Ascorbic Acid; Disease Models, Animal; Ferroptosis; Melanoma; Mice; Oxidative Stress; Sodium-Coupled Vitamin C Transporters

Whereas the prevalence of several cancer types is decreasing, skin malignancies are growing more common every year. Malignant melanoma is the most aggressive form of skin cancer with high metastatic capacity. In most cases, malignant melanoma shows acquired therapy resistance. We evaluated the ability of Ocoxin, a natural compound-based antioxidant and anti-inflammatory nutritional complement, to exert an antitumor effect in melanoma. To do so, the cytotoxicity of Ocoxin in a panel of BRAF-mutated murine and human melanoma cell lines was tested alone and in combination with BRAF inhibitor Vemurafenib. Our results revealed a potent cytotoxic effect of Ocoxin against melanoma cells and a synergic effect when combined with Vemurafenib, reducing viability and increasing apoptosis. Besides, Ocoxin interferes with the cell cycle, impairs the inherent and fibroblast-mediated melanoma cell migration, and reduces resistance to BRAF inhibition. Proteomic analysis revealed reduced tumor secretion of inflammatory factors Galectin-1, Osteopontin, CCL5, and CCL9 upon treatment with Ocoxin. Moreover, RNASeq showed that Ocoxin downregulated the cell cycle and proliferation-related genes. In vivo, Ocoxin reduced the number of lung metastasis of YUMM-1.7 melanoma cells. Therefore, Ocoxin arises as a good candidate for clinical trials analyzing the beneficial effects in patients suffering from this cutaneous malignancy.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Cancer-Associated Fibroblasts; Cell Line, Tumor; Drug Resistance, Neoplasm; Folic Acid; Humans; Melanoma; Melanoma, Cutaneous Malignant; Mice; Pantothenic Acid; Plant Extracts; Protein Kinase Inhibitors; Proteomics; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Vemurafenib; Vitamin B 12; Vitamin B 6; Zinc Sulfate

Vitamin C (VitC) is known to directly impair cancer cell growth in preclinical models, but there is little clinical evidence on its antitumoral efficacy. In addition, whether and how VitC modulates anticancer immune responses is mostly unknown. Here, we show that a fully competent immune system is required to maximize the antiproliferative effect of VitC in breast, colorectal, melanoma, and pancreatic murine tumors. High-dose VitC modulates infiltration of the tumor microenvironment by cells of the immune system and delays cancer growth in a T cell-dependent manner. VitC not only enhances the cytotoxic activity of adoptively transferred CD8 T cells but also cooperates with immune checkpoint therapy (ICT) in several cancer types. Combination of VitC and ICT can be curative in models of mismatch repair-deficient tumors with high mutational burden. This work provides a rationale for clinical trials combining ICT with high doses of VitC.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Immunotherapy; Melanoma; Mice; Tumor Microenvironment

Topics: Agaricales; Amides; Antineoplastic Agents; Cell Proliferation; Cell Survival; Cinnamates; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Melanoma; Molecular Docking Simulation; Molecular Structure; Monophenol Monooxygenase; Structure-Activity Relationship; Tumor Cells, Cultured

Dimeric cynnamoyl analogues (DCAs) with depigmenting activity have been developed. In this study, a role of diamide linkage chain length of DCAs as a tyrosinase inhibitor was investigated on tyrosinase inhibitory activity, antioxidative activity, hydrophobicity and anti-melanogenesis as well as structural characteristics and dipole moments based on density functional theory. DCAs with different diamide-link chain lengths (n = 2, 3, and 4) and various functional groups (m-coumaroyl, p-coumaroyl, isoferuloyl and feruloyl groups) were synthesized. DCAs with a diamide-link chain length of three indicated high inhibitory effect of melanin production on α-melanocyte stimulating hormone (α-MSH) stimulated B16F1 cells. Approach of p-hydroxyl group of DCAs to active site of tyrosinase, an important melanogenic enzyme, is interfered by addition of m-methoxy group. In structural modeling based on density functional theory, DCAs with a diamide-link chain length of three showed folded shapes, and they had lower dipole moment than with a diamide-link chain length of two or four. Thus, for the enhancement of the depigmenting activities of dimeric compounds, the diamide-link chain length is important. Our results provide an important index for the design of dimeric compounds with physiological activities.

Topics: Agaricales; Amides; Animals; Antioxidants; Cell Survival; Cinnamates; Density Functional Theory; Dimerization; Dose-Response Relationship, Drug; Melanoma; Mice; Molecular Structure; Monophenol Monooxygenase; Structure-Activity Relationship; Tumor Cells, Cultured

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in

Topics: Acetylation; Animals; Antioxidants; Apoptosis; Ascorbic Acid; Azepines; Biomarkers, Tumor; Cell Proliferation; Drug Combinations; Drug Synergism; Gene Expression Regulation, Neoplastic; Histones; Humans; Melanoma; Mice; Mice, Inbred C57BL; Mice, Nude; Protein Domains; Proteins; Triazoles; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The use of the well-known powerful antioxidant ascorbate has recently become more widespread in human medicine. Intravenous administration of high-dose ascorbate has been demonstrated to exert anticancer effects. It has resulted in effective cell death in vitro and inhibition of tumour growth in vivo. The aim of the current study was to evaluate the effects of high-dose ascorbate on canine melanoma in vitro. Four canine melanoma cell lines, UCDK9M1, UCDK9M3, UCDK9M4 and UCDK9M5 were treated with ascorbate for 2 hours at a range of millimolar concentrations (0-20 mM) to investigate the resulting effects on cell viability. All four canine melanoma cell lines exhibited reduced viability in a dose-dependent manner. Further investigation demonstrated that high-dose ascorbate induced apoptosis via the activation of Bax. These findings suggest that high-dose ascorbate has an anticancer effect on canine melanoma cell lines in vitro. With regard to clinical application, further in vivo investigation should be conducted.

Topics: Animals; Antineoplastic Agents; Apoptosis; Ascorbic Acid; Blotting, Western; Catalase; Cell Line, Tumor; Cell Survival; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Flow Cytometry; Melanoma

Topics: Adult; Aged; Ascorbic Acid; beta Carotene; Chocolate; Collagen; Curcuma; Diet, Healthy; Dietary Supplements; Female; Fibroblasts; Glycine max; Humans; Isoflavones; Linoleic Acid; Lutein; Lycopene; Melanoma; Middle Aged; Receptors, Calcitriol; Skin; Skin Aging; Skin Care; Sunlight; Tea; Vitamin D; Vitamin E

In the search for compounds which may inhibit the development of melanomas, a series of thiosemicarbazones has been investigated as possible inhibitors of the tyrosinase enzyme. The results showed that all the thiosemicarbazones tested exhibited significant inhibitory effects on the enzyme. Thiosemicarbazones Thio-1, Thio-2, Thio-3 and Thio-4 substituted with oxygenate moieties, were better inhibitors (IC

Topics: Agaricales; Enzyme Inhibitors; Humans; Levodopa; Melanins; Melanoma; Molecular Docking Simulation; Monophenol Monooxygenase; Thiosemicarbazones

Vitamin C has been used in complementary and alternative medicine for cancers regardless of its ineffectiveness in clinical trials and the paradoxical effects antioxidants have on cancer. Vitamin C was found to induce cytotoxicity against cancers. However, the mechanisms of action have not been fully elucidated, and the effects of vitamin C on human malignant melanoma have not been examined. This study revealed that vitamin C at millimolar concentrations significantly reduced the cell viability as well as invasiveness, and induced apoptosis in human malignant melanoma cells. Vitamin C displayed stronger cytotoxicity against the Vemurafenib-resistance cell line A2058 compared with SK-MEL-28. In contrast, vitamin C at micromolar concentrations promoted cell growth, migration and cell cycle progression, and protected against mitochondrial stress. Vemurafenib paradoxically activated the RAS-RAF-MEK-ERK signaling pathway in the Vemurafenib-resistant A2058, however, vitamin C abolished the activations. Vitamin C displayed synergistic cytotoxicity with Vemurafenib against the Vemurafenib-resistant A2058. In vivo assay suggested that lower dosage (equivalent to 0.5 g/70 kg) of vitamin C administered orally increased the melanoma growth. Therefore, vitamin C may exert pro- or anti-melanoma effect depending on concentration. The combination of vitamin C at high dosage and Vemurafenib is promising in overcoming the action of drug resistance.

Topics: Animals; Antineoplastic Agents; Antioxidants; Ascorbic Acid; Cell Cycle; Cell Line, Tumor; Cell Movement; Dose-Response Relationship, Drug; Drug Synergism; Humans; Indoles; Male; MAP Kinase Signaling System; Melanoma; Melanoma, Cutaneous Malignant; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Skin; Skin Neoplasms; Sulfonamides; Vemurafenib

A hypoxic tumor microenvironment is linked to poor prognosis. It promotes tumor cell dedifferentiation and metastasis and desensitizes tumor cells to type-I IFN, chemotherapy, and irradiation. The cytoplasmic immunoreceptor retinoic acid-inducible gene-I (RIG-I) is ubiquitously expressed in tumor cells and upon activation by 5'-triphosphate RNA (3pRNA) drives the induction of type I IFN and immunogenic cell death. Here, we analyzed the impact of hypoxia on the expression of RIG-I in various human and murine tumor and nonmalignant cell types and further investigated its function in hypoxic murine melanoma. 3pRNA-inducible RIG-I-expression was reduced in hypoxic melanoma cells compared with normoxic controls, a phenomenon that depended on the hypoxia-associated transcription factor HIF1α. Still, RIG-I functionality was conserved in hypoxic melanoma cells, whereas responsiveness to recombinant type-I IFN was abolished, due to hypoxia-induced loss of type I IFN receptor expression. Likewise, RIG-I activation in hypoxic melanoma cells, but not exposure to recombinant IFNα, provoked melanocyte antigen-specific CD8

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Line; Cell Line, Tumor; Female; Gene Knockout Techniques; Humans; Hypoxia; Immune Tolerance; Melanoma; Mice, Inbred C57BL; Reactive Oxygen Species; Receptors, Retinoic Acid; RNA; Spleen

Expression and function of Ten-eleven translocation (TET) enzymes, which initiate DNA demethylation by catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5 hmC) on methylated DNA, are frequently lost in malignant tissue. This ultimately results in lost expression of methylated tumor suppressor genes. Many malignancies, including melanoma, also aberrantly overexpress the oncogenic hypoxia inducible factor-1α (HIF-1α) transcription factor, however the association between HIF-1α and TET enzyme expression is largely uninvestigated. Interestingly, ascorbic acid, a critical cofactor for optimal TET enzyme function and normoxic regulation of HIF-1α protein stability, is frequently depleted in malignant tissue, and may further contribute to the malignant phenotype. In our studies, we found supplementation of WM9 human metastatic melanoma cells with ascorbic acid significantly increased 5 hmC content, which was abrogated by TET2 knockdown. Moreover, knockdown of HIF-1α increased TET2 gene and protein expression, and further augmented ascorbic acid-induced TET2 dependent 5-hydroxymethylation in both WM9 and T98G glioblastoma cells. Our data provides novel evidence that HIF-1α is involved in regulating TET expression and 5 hmC status of malignant cells. Furthermore, therapeutic intervention to inhibit HIF-1α in conjunction with adjuvant ascorbic acid may promote DNA demethylation and reexpression of critical tumor suppressor genes in malignant cells and warrants further investigation.

Topics: Ascorbic Acid; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Gene Expression Regulation, Enzymologic; Gene Silencing; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Melanoma; Proto-Oncogene Proteins; Tumor Cells, Cultured

Pharmacological levels of ascorbate have long been suggested as a potential treatment of cancer. However, we observed that EC50 of ascorbate was at a similar level for cultured healthy melanocytes and melanoma cells, suggesting a limit of pharmacological ascorbate in treating cancer. Loss of 5-hydroxymethylcytosine (5 hmC) is an epigenetic hallmark of cancer and ascorbate promotes 5 hmC generation by serving as a cofactor for TET methylcytosine dioxygenases. Our previous work demonstrated that ascorbate treatment at physiological level (100 μM) increased 5 hmC content in melanoma cells toward the level of healthy melanocytes. Here we show that 100 µM of ascorbate induced apoptosis in A2058 melanoma cells. RNA-seq analysis revealed that expression of the Clusterin (CLU) gene, which is related to apoptosis, was downregulated by ascorbate. The suppression of CLU was verified at transcript level in different melanoma cell lines, and at protein level in A2058 cells. The anti-apoptotic cytoplasmic CLU was decreased, while the pro-apoptotic nuclear CLU was largely maintained, after ascorbate treatment. These changes in CLU subcellular localization were also associated with Bax and caspases activation, Bcl-xL sequestration, and cytochrome c release. Taken together, this study establishes an impending therapeutic role of physiological ascorbate to potentiate apoptosis in melanoma.

Topics: Apoptosis; Ascorbic Acid; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Survival; Clusterin; Gene Expression Profiling; Gene Expression Regulation; Humans; Melanoma; Transcriptome

While surgery is the definitive treatment for early-stage melanoma, the current therapies against advanced melanoma do not yet provide an effective, long-lasting control of the lesions and a satisfactory impact on patient survival. Thus, research is also focused on novel treatments that could potentiate the current therapies. In the present study, we evaluated the effect of potassium ascorbate with ribose (PAR) treatment on the human melanoma cell line, A375, in 2D and 3D models. In the 2D model, in line with the current literature, the pharmacological treatment with PAR decreased cell proliferation and viability. In addition, an increase in Connexin 43 mRNA and protein was observed. This novel finding was confirmed in PAR-treated melanoma cells cultured in 3D, where an increase in functional gap junctions and a higher spheroid compactness were observed. Moreover, in the 3D model, a remarkable decrease in the size and volume of spheroids was observed, further supporting the treatment efficacy observed in the 2D model. In conclusion, our results suggest that PAR could be used as a safe adjuvant approach in support to conventional therapies for the treatment of melanoma.

Topics: Antineoplastic Agents; Ascorbic Acid; Cell Line, Tumor; Cell Proliferation; Cell Survival; Connexin 43; Humans; Melanoma; Microscopy, Electron, Scanning; Potassium; Ribose; Spheroids, Cellular

Accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in malignant tissue is known to contribute to oncogenic progression and is inversely associated with patient survival. Ascorbic acid (AA) depletion in malignant tissue may contribute to aberrant normoxic activity of HIF-1α. While AA supplementation has been shown to attenuate HIF-1α function in malignant melanoma, the use of dehydroascorbic acid (DHA) as a therapeutic means to increase intracellular AA and modulate HIF-1α function is yet to be evaluated. Here we compared the ability of AA and DHA to increase intracellular vitamin C content and decrease the malignant potential of human melanoma by reducing the activity of HIF-1α.. HIF-1α protein accumulation was evaluated by western blot and transcriptional activity was evaluated by reporter gene assay using a HIF-1 HRE-luciferase plasmid. Protein expressions and subcellular localizations of vitamin C transporters were evaluated by western blot and confocal imaging. Intracellular vitamin C content following AA, ascorbate 2-phosphate (A2P), or DHA supplementation was determined using a vitamin C assay. Malignant potential was accessed using a 3D spheroid Matrigel invasion assay. Data was analyzed by One or Two-way ANOVA with Tukey's multiple comparisons test as appropriate with p<0.05 considered significant.. Melanoma cells expressed both sodium dependent vitamin C (SVCT) and glucose (GLUT) transporters for AA and DHA transport respectively, however advanced melanomas responded favorably to AA, but not DHA. Physiological glucose conditions significantly impaired intracellular vitamin C accumulation following DHA treatment. Consequently, A2P and AA, but not DHA treated cells demonstrated lower HIF-1α protein expression and activity, and reduced malignant potential. The ability of AA to regulate HIF-1α was dependent on SVCT2 function and SVCT2 was not significantly inhibited at pH representative of the tumor microenvironment.. The use of ascorbic acid as an adjuvant cancer therapy remains under investigated. While AA and A2P were capable of modulating HIF-1α protein accumulation/activity, DHA supplementation resulted in minimal intracellular vitamin C activity with decreased ability to inhibit HIF-1α activity and malignant potential in advanced melanoma. Restoring AA dependent regulation of HIF-1α in malignant cells may prove beneficial in reducing chemotherapy resistance and improving treatment outcomes.

Topics: Ascorbic Acid; Biological Transport; Cell Line; Cell Line, Tumor; Dehydroascorbic Acid; Glucose; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Melanoma; Sodium; Transcription, Genetic; Tumor Microenvironment

In our searching for novel tyrosinase inhibitors from natural sources, (S)-N-trans-feruloyloctopamine isolated from garlic skin was found to be a potential mushroom tyrosinase inhibitor. Here, we examined the effects of the potential tyrosinase inhibitor in B16F10 cells on intracellular melanin contents, cytotoxicity, and the signaling mechanism involved in the expression of tyrosinase. The results showed the inhibitor displayed little or no cytotoxicity at all concentrations examined and decreased the relative melanin contents in a dose-dependent manner in the α-MSH-stimulated B16F10 cells. Real-time PCR and Western blot analysis showed that it inhibits melanogenesis signaling by down-regulates mRNA and protein expression levels of tyrosinase, which leads to a lower melanin contents. These results suggested that (S)-N-trans-feruloyloctopamine was an ideal tyrosinase inhibitor, and could be used in food and medical industry.

Topics: Agaricales; Animals; Cell Line, Tumor; Coumaric Acids; Dose-Response Relationship, Drug; Enzyme Inhibitors; Garlic; Gene Expression Regulation, Enzymologic; Melanins; Melanoma; Mice; Monophenol Monooxygenase; Octopamine; Structure-Activity Relationship

Alpha-tocopheryl succinate (αTOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although αTOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 μM αTOS/20 μM AA/0.2 μM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention.

Topics: alpha-Tocopherol; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Ascorbic Acid; Cell Line, Tumor; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Drug Synergism; Humans; Killer Cells, Natural; Melanoma; Oxidants; Oxidative Stress; Signal Transduction; Skin Neoplasms; Vitamin K 3; Vitamins

Hypoxia inducible factor-1 alpha (HIF-1α) is thought to play a role in melanoma carcinogenesis. Posttranslational regulation of HIF-1α is dependent on Prolyl hydroxylase (PHD 1-3) and Factor Inhibiting HIF (FIH) hydroxylase enzymes, which require ascorbic acid as a co-factor for optimal function. Depleted intra-tumoral ascorbic acid may thus play a role in the loss of HIF-1α regulation in melanoma. These studies assess the ability of ascorbic acid to reduce HIF-1α protein and transcriptional activity in metastatic melanoma and reduce its invasive potential.. HIF-1α protein was evaluated by western blot, while transcriptional activity was measured by HIF-1 HRE-luciferase reporter gene activity. Melanoma cells were treated with ascorbic acid (AA) and ascorbate 2-phosphate (A2P) to assess their ability to reduce HIF-1α accumulation and activity. siRNA was used to deplete cellular PHD2 in order to evaluate this effect on AA's ability to lower HIF-1α levels. A2P's effect on invasive activity was measured by the Matrigel invasion assay. Data was analyzed by One-way ANOVA with Tukey's multiple comparisons test, or Student-T test as appropriate, with p < .05 considered significant.. Supplementation with both AA and A2P antagonized normoxic as well as cobalt chloride- and PHD inhibitor ethyl 3, 4-dihydroxybenzoate induced HIF-1α protein stabilization and transcriptional activity. Knockdown of the PHD2 isoform with siRNA did not impede the ability of AA to reduce normoxic HIF-1α protein. Additionally, reducing HIF-1α levels with A2P resulted in a significant reduction in the ability of the melanoma cells to invade through Matrigel.. These studies suggest a positive role for AA in regulating HIF-1α in melanoma by demonstrating that supplementation with either AA, or its oxidation-resistant analog A2P, effectively reduces HIF-1α protein and transcriptional activity in metastatic melanoma cells. Our data, while supporting the function of AA as a necessary cofactor for PHD and likely FIH activity, also suggests a potential non-PHD/FIH role for AA in HIF-1α regulation by its continued ability to reduce HIF-1α in the presence of PHD inhibition. The use of the oxidation-resistant AA analog, A2P, to reduce the ability of HIF-1α to promote malignant progression in melanoma cells and enhance their response to therapy warrants further investigation.

Topics: Ascorbic Acid; Cell Hypoxia; Cell Line, Tumor; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Melanoma; Neoplasm Metastasis; Protein Stability; Transcription, Genetic

Cutaneous melanoma incidence has been increasing during the last few years, and diet has been suggested as one of the lifestyle factors responsible for this increase. Since antioxidant nutrients such as ascorbic acid might prevent skin carcinogenesis, we investigated the risk of cutaneous melanoma related to vitamin C intake in a population-based case-control study in Northern Italy based on 380 melanoma patients and 719 matched controls, to whom we administered a semiquantitative food-frequency questionnaire. After adjusting for potential confounders, odds ratio of melanoma were 0.86 (95 % confidence interval 0.65 - 1.15) and 0.59 (95 % confidence interval 0.37 - 0.94) in the intermediate and highest categories of vitamin C dietary intake respectively, compared with the bottom one. The association between vitamin C and decreased risk persisted after adjustment for some potential confounders. In age- and gender-stratified analyses, this association was seen in young females (< 60 years old), and was found to be enhanced in subjects with phototypes II and III. These results suggest a possible protective activity of vitamin C intake against cutaneous melanoma in specific subgroups of this population of Northern Italy.

Topics: Age Distribution; Antioxidants; Ascorbic Acid; Case-Control Studies; Diet; Feeding Behavior; Female; Humans; Italy; Male; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Odds Ratio; Risk Factors; Sex Distribution; Skin Neoplasms; Surveys and Questionnaires

Sulfonamides of halogenated bacteriochlorins bearing Cl or F substituents in the ortho positions of the phenyl rings have adequate properties for photodynamic therapy, including strong absorption in the near-infrared (λ(max) ≈ 750 nm, ε ≈ 10(5) M(-1) cm(-1)), controlled photodecomposition, large cellular uptake, intracellular localization in the endoplasmic reticulum, low cytotoxicity, and high phototoxicity against A549 and S91 cells. The roles of type I and type II photochemical processes are assessed by singlet oxygen luminescence and intracellular hydroxyl radical detection. Phototoxicity of halogenated sulfonamide bacteriochlorins does not correlate with singlet oxygen quantum yields and must be mediated both by electron transfer (superoxide ion, hydroxyl radicals) and by energy transfer (singlet oxygen). The photodynamic efficacy is enhanced when cellular death is induced by both singlet oxygen and hydroxyl radicals.

Topics: Adenocarcinoma; Antioxidants; Ascorbic Acid; Cell Survival; Energy Transfer; Fluorescence; Humans; Hydroxyl Radical; In Vitro Techniques; Lung Neoplasms; Melanoma; Photochemotherapy; Photosensitizing Agents; Porphyrins; Reactive Oxygen Species; Singlet Oxygen; Tumor Cells, Cultured

Ascorbic acid (AA) has been well known as a skin whitening agent, although attempts have been made to evaluate its protective role against ultraviolet (UV)-induced skin hyperpigmentation or increased melanin production. While melanogenesis is a defense mechanism of the skin against UV irradiation, melanin overproduction may also contribute to melanoma initiation. UVA might play a role in melanogenesis through promoting oxidative stress, which occurs as the result of increased formation of oxidants and/or reactive nitrogen species (RNS) including nitric oxide (NO). Therefore, we investigated the antimelanogenic effect of AA (7.5-120 μM) in association with its inhibitory effect on UVA-induced oxidant formation, NO production through endothelial and inducible NO synthases (eNOS and iNOS) activation and impairment of antioxidant defense using G361 human melanoma cells. Our study demonstrated a comparable ability of AA with that of kojic acid, a well-known tyrosinase inhibitor in inhibiting mushroom tyrosinase. Melanin content was reduced by AA, but neither tyrosinase activity nor mRNA levels were reduced by AA at non-cytotoxic concentrations in UVA-irradiated G361 cells. AA was shown to inhibit UVA-mediated catalase (CAT) inactivation, glutathione (GSH) depletion, oxidant formation and NO production through suppression of eNOS and iNOS mRNA. We report herein that AA can protect against UVA-dependent melanogenesis possibly through the improvement of antioxidant defense capacity and inhibition of NO production through down-regulation of eNOS and iNOS mRNA.

Topics: Antioxidants; Ascorbic Acid; Catalase; Cell Line, Tumor; Cell Survival; Dermatologic Agents; Enzyme Inhibitors; Fungal Proteins; Gene Expression Regulation, Enzymologic; Glutathione; Humans; Melanins; Melanocytes; Melanoma; Monophenol Monooxygenase; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; RNA, Messenger; Skin Pigmentation; Ultraviolet Rays

Ascorbic acid, a potential antioxidant, is known to inhibit melanogenesis. However, there are conflicting findings that ascorbic acid has very low stability and acts as a pro-oxidant, eventually increasing proliferation and melanin content in melanoma cells. In the present study, we explored the effects of ascorbic acid on the activity and expression of tyrosinase and melanin pigmentation in the presence and absence of α-melanocyte-stimulating hormone (α-MSH) using B16F10 melanoma cells. The mechanism by which ascorbic acid stimulated the expression of tyrosinase was also investigated. No inhibitory effect on melanin content was observed in ascorbic acid-treated cells, regardless of the presence of α-MSH. Ascorbic acid stimulated the activity and expression of tyrosinase and increased the expression of melanogenic regulatory factors, such as tyrosinase-related protein-1 (TRP-1), dihydroxyphenylalaminechrome tautomerase (TRP-2), and microphthalmia-associated transcription factor (MITF). Ascorbic acid also induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). The inhibition of p38 MAPK pathway by SB203580 led to the suppression of tyrosinase, TRP-1, and TRP-2 expression in cells treated with ascorbic acid. Combined treatment with N-acetyl-L: -cysteine and/or desferrioxamine mesylate attenuated the stimulating effect of ascorbic acid on tyrosinase activation in the cells. Collectively, ascorbic acid stimulates tyrosinase activity and expression in B16F10 cells via activation of p38 MAPK signaling and subsequent up-regulation of MITF, tyrosinase, and TRP expression.

Topics: alpha-MSH; Animals; Anti-Inflammatory Agents, Non-Steroidal; Ascorbic Acid; Deferoxamine; Disease Models, Animal; Enzyme Activation; Humans; Imidazoles; Intramolecular Oxidoreductases; MAP Kinase Signaling System; Melanins; Melanoma; Melanoma, Experimental; Mice; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Oxidoreductases; Pyridines

Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.

Topics: Ascorbic Acid; Biological Transport; Biotinylation; Cell Membrane; Histidine; Humans; Hydrogen-Ion Concentration; Kidney; Kinetics; Melanoma; Membrane Proteins; Mutagenesis, Site-Directed; Organic Anion Transporters, Sodium-Dependent; Protein Conformation; Sodium; Sodium-Coupled Vitamin C Transporters; Subcellular Fractions; Symporters

Irradiation with ultraviolet-A (UVA) ray at doses of 20-100 J/cm(2) diminished the cell viability of human keratinocytes HaCaT and human melanoma cells HMV-II, both of which were protected by pre-irradiational administration with the ascorbic acid (Asc) derivative, VC-IP (2,3,5,6-O-tetra-2'-hexyldecanoyl-L-ascorbic acid; vitamin C-isopalmityl tetraester), which is the first lipoidic-liquiform pro-vitamin C by itself that is materialized by esterization of all four intramolecular hydroxyl groups of an Asc molecule with branched chain fatty groups, resulting in molecular fluidity higher than that of the corresponding straight chains. Irradiation with UVA to HaCaT keratinocytes was shown to cause the formation of 8-hydroxydeoxyguanosine (8-OHdG), translocation of phosphatidylserine in the inner layer into the outer layer of cell membrane, and lowering of a mitochondrial membrane potential, all of which were repressed by pre-irradiational administration with VC-IP. Expression of p53 gene, another hallmark of UV-induced DNA damages, was promoted by UVA irradiation to the keratinocytes but also repressed by VC-IP. Administration with VC-IP of 10-50 microM to human fibroblasts NHDF achieved the enhancement of collagen synthesis, repression of matrix metalloprotease-2/9 activity, and increasing of intracellular Asc contents more markedly than that with Asc itself of the same concentrations. Thus UVA-induced diverse harmful effects could be prevented by VC-IP, which was suggested to ensue intrinsically from the persistent enrichment of intracellular Asc, through esterolytic conversion of VC-IP to a free-form Asc molecule, resulting in relief to UVA-caused oxidative stress.

Topics: Ascorbic Acid; Cell Line; Cell Survival; Collagen; Cytoprotection; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Glycolipids; Humans; Keratinocytes; Matrix Metalloproteinase Inhibitors; Melanoma; Skin; Tumor Suppressor Protein p53; Ultraviolet Rays

The cell ability of tumor cells to tolerate stress conditions is a typical feature of solid tumors. In particular, the resistance to oxidative stress of melanoma cells likely contributes to their intrinsic drug resistance. In an attempt to develop novel strategies for overcoming the mechanisms of cellular protection against oxidative stress, in this study we have explored the efficacy of the combination of two prooxidant agents in two human melanoma cell clones. The selected clones are characterized by a marked difference in expression of gamma-glutamyltransferase, which is known to produce a persistent low level of oxidative stress resulting in the stimulation of protective systems. The gamma-glutamyltransferase-overexpressing clone exhibited a low susceptibility to arsenic trioxide-induced apoptosis, associated with low reactive oxygen species induction and increased catalase activity. The combination of arsenic trioxide with subtoxic concentrations of ascorbic acid resulted in a sensitization to apoptotic cell death. The expression of protective mechanisms, in particular catalase activity, accounted for the behavior of the resistant clone. The sensitization achieved by the combination was associated with a cellular response involving the ASK1/p38 axis, which is implicated in the regulation of catalase expression and the activation of apoptotic signals. In conclusion, the results of our study provide evidence that a rational combination of prooxidant agents may be effective in overcoming cellular tolerance to oxidative stress.

Topics: Apoptosis; Arsenic Trioxide; Arsenicals; Ascorbic Acid; Catalase; Cell Line, Tumor; Cytoprotection; Drug Resistance, Neoplasm; Enzyme Activation; gamma-Glutamyltransferase; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; MAP Kinase Kinase Kinase 5; Melanoma; Oxidative Stress; Oxides

The extracellular matrix (ECM) that gives tissue its structural integrity is remodeled in skin aging/photoaging and cancer via the increased expression/activities of matrixmetalloproteinases (MMP), inhibition of the tissue inhibitors of matrix metalloproteinases (TIMP), or inhibition of collagen synthesis. Transforming growth factor-beta (TGF-beta), a predominant regulator of the ECM, is inhibited in aging/photoaging and stimulated in carcinogenesis. P. leucotomos (fern) extract has potential to counteract these alterations via its antioxidant, anti-inflammatory and photoprotective properties. The goal of this research was to determine the efficacy of P. leucotomos to (a) directly inhibit MMP-1, 2, 3, and 9 activities, (b) inhibit MMP-2, and stimulate TIMPs, fibrillar collagens and TGF-beta in non-irradiated or ultraviolet (UV) radiated fibroblasts, and (c) inhibit MMPs and TGF-beta, and stimulate TIMPs in melanoma cells. To this purpose, we examined the direct effect of P. leucotomos (0-1%) on MMPs' activities, and its effects on the expression (protein and/or transcription levels) of (1) MMPs and TIMPs in dermal fibroblasts, and melanoma cells, (2) TGF-beta in non-irradiated, UVA (2.5 J/cm2) or UVB (2.5 mJ/cm2) irradiated fibroblasts, and melanoma cells, and (3) types I, III, and V collagen in non-irradiated or UV irradiated fibroblasts. P. leucotomos directly inhibited the activities of MMPs as well as the expression of MMPs in fibroblasts, and melanoma cells while stimulating the expression of TIMPs in these cells. P. leucotomos stimulated types I, III, and V collagen in non-irradiated fibroblasts, and types I and V collagen in UV radiated fibroblasts. P. leucotomos had predominant stimulatory effects on TGF-beta expression in non-irradiated or UV radiated fibroblasts, and inhibited TGF-beta expression in melanoma cells. The effects of P. leucotomos were largely similar to that of ascorbic acid. P. leucotomos demonstrated dual protective effects on the ECM via its inhibition of the ECM proteolytic enzymes and the stimulation of the structural ECM collagens. The effects of P. leucotomos on fibroblasts and melanoma cells may be partly via its cell-specific regulation of TGF-beta expression and partly via its antioxidant property. The intake or topical application of P. leucotomos may be beneficial to skin health, in aging and cancer prevention or treatment.

Topics: Antioxidants; Ascorbic Acid; Cells, Cultured; Cytoprotection; Extracellular Matrix; Fibrillar Collagens; Fibroblasts; Gene Expression Regulation; Humans; Infant, Newborn; Matrix Metalloproteinases; Melanoma; Plant Extracts; Polypodium; Skin Aging; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Ultraviolet Rays

Ascorbate has dose-dependent inverse effects on cancer cells growth and expression of matrixmetalloproteinases (MMP) and transforming growth factor-beta (TGF-beta), which regulate extracellular matrix (ECM) remodeling. We examined melanoma cell viability and ECM remodeling mechanisms of ascorbate and its modulation by an extract from Polypodium leucotomos (PL) (a fern) via the regulation of apoptosis, heat-shock proteins (HSPs), MMP-1 or tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) that inhibits MMP-1. The dose-dependent regulation of cell viability/proliferation by ascorbate was associated with inverse regulation of apoptosis and stimulation of HSPs at growth-inhibitory concentrations. PL antagonized the stimulation of MMP-1, TGF-beta and HSPs by a growth-inhibitory ascorbate dose and stimulated the expression of TIMP-1, while maintaining growth inhibition. We infer that a combination of ascorbate with PL is beneficial to cancer management via the simultaneous inhibition of cell growth and expression of MMP-1, TGF-beta and HSPs, and furthermore, the stimulation of TIMP-1.

Topics: Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Ascorbic Acid; Caspase 3; Cell Proliferation; Cells, Cultured; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Heat-Shock Proteins; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase Inhibitors; Melanoma; Plant Extracts; Polypodium; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Tumor Cells, Cultured

Vitamin C plays a crucial role in the suppression of proliferation of several types of cancer. Over-expression of cyclooxygenase (COX)-2 and type I insulin-like growth factor (IGF) receptor are important for proliferation and protection from apoptosis in malignancies. However, its specific mechanisms, especially the interaction between COX-2 expression and IGF-I axis mediated by vitamin C, remain yet to be clarified. Therefore, we investigated the effects of vitamin C on the proliferation of melanoma cells via the modulation of COX-2 expression and IGF-I axis. As a result, we found that 1.0 mM vitamin C inhibits the proliferation of SK-MEL-2 without induction of apoptosis. At that moment, IGF-II production was decreased, followed by the inhibition of COX-2 activity. IGF-IR expression was also down-regulated by vitamin C treatment. It coincided with the result from the inhibition of COX-2 by NS-398 and COX-2 siRNA. In addition, the decreased IGF-IR expression by vitamin C was restored by the treatment of recombinant prostaglandin E2. Finally, we determined whether the signal pathway would be involved in vitamin C-induced IGF-II and IGF-IR down-regulation. When the cells were exposed to SB203580, a specific inhibitor of p38 MAPK, COX-2 expression was dramatically recovered. In addition, phosphorylated p38 MAPK was increased after vitamin C treatment. Taken together, vitamin C suppresses proliferation of the human melanoma cell line SK-MEL2 via the down-regulation of IGF-II production and IGF-IR expression, which is followed by the activation of p38 MAPK and the inhibition of COX-2 expression.

Topics: Animals; Ascorbic Acid; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Enzyme Activation; Enzyme Inhibitors; Humans; Imidazoles; Insulin-Like Growth Factor II; Melanoma; p38 Mitogen-Activated Protein Kinases; Pyridines; Receptor, IGF Type 1; RNA, Small Interfering; Vitamins

We investigated the inhibitory effects of a novel amphiphilic ascorbic derivative, disodium isostearyl 2-O-L-ascorbyl phosphate (VCP-IS-2Na), synthesized from a hydrophilic ascorbic derivative, sodium-2-O-L-ascorbyl phosphate (VCP-Na), on melanogenesis in cultured human melanoma cells, normal human melanocytes, and three-dimensional cultured human skin models. Melanin synthesis in melanoma cells treated with VCP-IS-2Na at 300 muM and melanocytes treated with VCP-IS-2Na at 100 muM decreased to 23% and 52% of that in non-treated cells, respectively, and the cell viability was not affected. VCP-IS-2Na also significantly suppressed the cellular tyrosinase activity of melanoma cells and melanocytes. Melanin synthesis in human skin models was evaluated by macro- and microscopic observations of its pigmentation and quantitative measurements of melanin. Treatment of the human skin models with 1.0% VCP-IS-2Na did not inhibit cell viability, while melanin synthesis was decreased to 21% of that in the control. In contrast, L-ascorbic acid (VC) and VCP-Na did not seem to inhibit melanin synthesis and cellular tyrosinase activity. These results indicate that VCP-IS-2Na may be an effective whitening agent for the skin, and we expect the application of VCP-IS-2Na in the cosmetic industry.

Topics: Ascorbic Acid; Cell Line, Tumor; Cell Proliferation; Humans; Indicators and Reagents; Magnetic Resonance Spectroscopy; Melanins; Melanoma; Monophenol Monooxygenase; Skin; Spectrometry, Mass, Fast Atom Bombardment; Spectrophotometry, Ultraviolet; Tetrazolium Salts; Thiazoles

The extracellular gamma-glutamyltransferase-mediated metabolism of glutathione has been implicated in prooxidant events which may have impact on cellular functions including drug resistance. This study was performed in two GGT-transfected melanoma clones to explore the hypothesis that GGT expression in tumour cells is implicated in modulation of cell behaviour under stress conditions. Our results show that GGT-overexpression in melanoma cells was associated with resistance to oxidative stress produced by prooxidant agents such as hydrogen peroxide and ascorbic acid. In GGT-overexpressing cells, ability to tolerate oxidative stress was evidenced by the presence of a moderate level of ROS and lack of DNA damage response following treatment with H(2)O(2). Cellular response to oxidative stress induced by ascorbic acid was detectable only in the clone with low GGT activity which also exhibited an increased susceptibility to apoptosis. The increased resistance of the GGT-overexpressing clone was not related to intracellular GSH content but rather to the increased expression of catalase and to a reduced efficiency of iron-mediated formation of toxic free radicals. Taken together, these findings are consistent with a contribution of GGT in the mechanisms of drug resistance, because induction of oxidative stress is a relevant event in the apoptotic response to cytotoxic agents.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Blotting, Western; Catalase; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drug Resistance, Neoplasm; gamma-Glutamyltransferase; Humans; Hydrogen Peroxide; Melanoma; Mitochondria; Oxidative Stress; Reactive Oxygen Species

We characterized the human Na(+)-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na(+)-dependent transporter. The properties of SVCT2 are modulated by Ca(2+)/Mg(2+) and a reciprocal functional interaction between Na(+) and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na(+) increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K(m) without affecting the V(max), thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na(+) cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na(+):ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na(+):ascorbic acid:Na(+). However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca(2+)/Mg(2+) for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V(max) without affecting the transport K(m) or the Na(+) cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca(2+)/Mg(2+). At least three active states can be envisioned, including a low affinity conformation at Na(+) concentrations below 20 mM and two high affinity conformations at elevated Na(+) concentrations whose Na(+) cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca(2+)/Mg(2+)-dependent transporter.

Topics: Amino Acid Sequence; Ascorbic Acid; Calcium; Cations; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Kinetics; Magnesium; Melanoma; Models, Biological; Molecular Sequence Data; Organic Anion Transporters, Sodium-Dependent; Sequence Homology, Amino Acid; Sodium; Sodium-Coupled Vitamin C Transporters; Symporters

Cancer is associated with increased cell growth, and expression of matrix metalloproteinases (MMPs) and transforming growth factor-beta (TGF-beta). The dose-dependent effects of ascorbate (Vitamin C) on cancer cell growth, and expression of MMPs and TGF-beta were examined. Renal-adenocarcinoma, melanoma and mammary cancer cells were dosed with 0-100mM ascorbate and examined for cell survival or proliferation, and expression of MMP-1, MMP-2 and TGF-beta at protein and/or mRNA levels. The lower concentrations of ascorbate significantly inhibited cancer cell viability while stimulating MMPs and TGF-beta expression, indicating elimination of cancer cells with damage to the extracellular matrix (ECM). Conversely, ascorbate at higher concentrations dramatically stimulated cell proliferation and inhibited MMPs and TGF-beta expression, implicating growth and ECM advantage.

Topics: Adenocarcinoma; Antioxidants; Ascorbic Acid; Breast Neoplasms; Cell Survival; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Humans; Kidney Neoplasms; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Melanoma; RNA, Messenger; Skin Neoplasms; Transforming Growth Factor beta1; Tumor Cells, Cultured

Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.

Topics: Apoptosis; Ascorbic Acid; Calcium; Caspase 3; Cell Proliferation; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Flow Cytometry; G1 Phase; Humans; Intracellular Signaling Peptides and Proteins; Melanoma; Membrane Potential, Mitochondrial; Oncogene Proteins; Reactive Oxygen Species; S Phase; Skin Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Sodium ascorbate (vitamin C) has a reputation for inconsistent effects upon malignant tumor cells, which vary from growth stimulation to apoptosis induction. Melanoma cells were found to be more susceptible to vitamin C toxicity than any other tumor cells. The present study has shown that sodium ascorbate decreases cellular iron uptake by melanoma cells in a dose- and time-dependent fashion, indicating that intracellular iron levels may be a critical factor in sodium ascorbate-induced apoptosis. Indeed, sodium ascorbate-induced apoptosis is enhanced by the iron chelator, desferrioxamine (DFO) while it is inhibited by the iron donor, ferric ammonium citrate (FAC). Moreover, the inhibitory effects of sodium ascorbate on intracellular iron levels are blocked by addition of transferrin, suggesting that transferrin receptor (TfR) dependent pathway of iron uptake may be regulated by sodium ascorbate. Cells exposed to sodium ascorbate demonstrated down-regulation of TfR expression and this precedes sodium ascorbate-induced apoptosis. Taken together, sodium ascorbate-mediated apoptosis appears to be initiated by a reduction of TfR expression, resulting in a down-regulation of iron uptake followed by an induction of apoptosis. This study demonstrates the specific mechanism of sodium ascorbate-induced apoptosis and these findings support future clinical trial of sodium ascorbate in the prevention of human melanoma relapse.

Topics: Animals; Antioxidants; Apoptosis; Ascorbic Acid; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Iron; Melanoma; Mice; Receptors, Transferrin; RNA Processing, Post-Transcriptional; Skin Neoplasms

The combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) has recently been proposed as a novel cancer therapy. However, the mechanism underlying the cytotoxic effect involved is substantially unknown. Here, we show that IAA/HRP treatment induces apoptosis in G361 human melanoma cells, whereas IAA or HRP alone have no effect. It is known that IAA produces free radicals when oxidized by HRP. Because oxidative stress could induce apoptosis, we measured the production of free radicals at varying concentrations of IAA and HRP. Our results show that IAA/HRP produces free radicals in a dose-dependent manner, which are suppressed by ascorbic acid or (-)-epigallocatechin gallate (EGCG). Furthermore, antioxidants prevent IAA/HRP-induced apoptosis, indicating that the IAA/HRP-produced free radicals play an important role in the apoptotic process. In addition, IAA/HRP was observed to activate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), which are almost completely blocked by antioxidants. We further investigated the IAA/HRP-mediated apoptotic pathways, and found that IAA/HRP activates caspase-8 and caspase-9, leading to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. These events were also blocked by antioxidants, such as ascorbic acid or EGCG. Thus, we propose that IAA/HRP-induced free radicals lead to the apoptosis of human melanoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Caspases; Catechin; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Free Radicals; Horseradish Peroxidase; Humans; Indoleacetic Acids; JNK Mitogen-Activated Protein Kinases; Melanoma; Mitochondria; Mitogen-Activated Protein Kinases; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prodrugs; Proteins

Adequate cellular transport of ascorbic acid (AA) and its oxidation product dehydroascorbate (DHA) is assured through specific carriers. It was shown that vitamin C is taken up as DHA by most cell types, including cancer cells, via the facilitative GLUT transporters. Thus, AA oxidation to DHA can be considered a mechanism favoring vitamin C uptake and intracellular accumulation. We have investigated whether such an AA-oxidizing action might be provided by plasma membrane gamma-glutamyltransferase (GGT), previously shown to function as an autocrine source of prooxidants. The process was studied using two distinct human metastatic melanoma clones. It was observed that the Me665/2/60 clone, expressing high levels of membrane GGT activity, was capable of effecting the oxidation of extracellular AA, accompanied by a marked increase of intracellular AA levels. The phenomenon was not observed with Me665/2/21 cells, possessing only traces of membrane GGT. On the other hand, AA oxidation and stimulation of cellular uptake were indeed observed after transfection of 2/21 cells with cDNA coding for GGT. The mechanism of GGT-mediated AA oxidation was investigated in acellular systems, including GGT and its substrate glutathione. The process was observed in the presence of redox-active chelated iron(II) and of transferrin or ferritin, i.e., two physiological iron sources. Thus, membrane GGT activity-often expressed at high levels in human malignancies-can oxidize extracellular AA and promote its uptake efficiently.

Topics: Ascorbic Acid; Biological Transport; Cell Line, Tumor; Cell Membrane; gamma-Glutamyltransferase; Glutathione; Humans; Iron Chelating Agents; Melanoma; Oxidation-Reduction; Skin Neoplasms; Transfection; Transferrin

Within the two Nurses' Health Study cohorts of US women, we examined whether higher intakes of vitamin C, vitamin E, retinol, or individual tocopherols or carotenoids are associated with a lower risk of melanoma. We confirmed 414 cases of invasive melanoma among over 162,000 Caucasian women aged 25-77 years during more than 1.6 million person-years of follow-up. Diet was measured every 4 years with a food frequency questionnaire and supplement use was reported every 2 years. Several measures of sun sensitivity were assessed and included in proportional hazards models. We found that vitamins A, C, E and their individual components were not associated with a lower risk of melanoma. Only retinol intake from foods plus supplements appeared protective within a subgroup of women who were otherwise at low risk based on nondietary factors (relative risk (RR)=0.39, 95% confidence interval (CI) 0.22-0.71 for >/=1,800 vs 400 microg day(-1), P for linear trend=0.01). Contrary to expectation, we observed higher risks of melanoma with greater intakes of vitamin C from food only (RR=1.43, 95% CI 1.01-2.00 for >/=175 vs <90 mg day(-1), P for linear trend=0.05) and a significant positive dose-response with frequency of orange juice consumption (P=0.008). Further research is needed to determine whether another component in foods such as orange juice may contribute to an increase in risk.

Topics: Adult; Aged; Ascorbic Acid; Boston; Cohort Studies; Diet; Female; Follow-Up Studies; Humans; Incidence; Melanoma; Middle Aged; Neoplasm Invasiveness; Risk Assessment; Risk Factors; Time Factors; Vitamin A; Vitamin E; White People

Antioxidants such as vitamins C and E have been reported to inhibit the progression of ultraviolet (UV) radiation-induced pigmentation in the skin of hairless mice. However, little is known of the lightening effect of proanthocyanidin, a powerful polyphenolic antioxidant, on UV-induced pigmentation of the skin. We investigated the lightening effect of oral administration of a proanthocyanidin-rich grape seed extract (GSE) using guinea pigs with UV-induced pigmentation. These pigmented guinea pigs were fed diets containing 1% GSE or 1% vitamin C (w/w) for 8 weeks. GSE-feeding had an apparent lightening effect on the guinea pigs' pigmented skin. Histologic evaluation demonstrated a decrease in the number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes as well as 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive, Ki-67-positive, proliferating cell nuclear antigen (PCNA)-positive melanin-containing cells in the basal epidermal layer of the UV-irradiated skin in GSE-fed guinea pigs. In contrast, these parameters did not change in the skin of vitamin C-fed or control guinea pigs. GSE inhibited the activity of mushroom tyrosinase and also inhibited melanogenesis without inhibiting the growth of cultured B16 mouse melanoma cells. In conclusion, we demonstrated that oral administration of GSE is effective in lightening the UV-induced pigmentation of guinea pig skin. This effect may be related to the inhibition of melanin synthesis by tyrosinase in melanocytes and the reactive oxygen species (ROS)-related proliferation of melanocytes.

Topics: Administration, Oral; Animals; Antioxidants; Ascorbic Acid; Deoxyadenosines; Dihydroxyphenylalanine; Guinea Pigs; Ki-67 Antigen; Melanocytes; Melanoma; Mice; Monophenol Monooxygenase; Proanthocyanidins; Proliferating Cell Nuclear Antigen; Reactive Oxygen Species; Skin; Skin Pigmentation; Tumor Cells, Cultured; Ultraviolet Rays; Vitis

We recently reported that interleukin-18 (IL-18) is highly expressed in malignant skin tumours such as melanomas, and may play a key role in the malignancy of such tumours. This study was designed to investigate the mechanisms of IL-18 regulation by vitamin C in B16F10 murine melanoma cells. Cells were treated with vitamin C, and the expression of IL-18 was measured by reverse transcription-polymerase chain reaction and intracellular flow cytometry analysis. Decreased IL-18 production and a significant reduction in IL-18 mRNA transcript were detected in cells treated with vitamin C. The effect of vitamin C treatment was blocked by the antioxidant N-acetyl-L-cysteine, suggesting that vitamin C affects IL-18 expression by up-regulating intracellular reactive oxygen intermediate (ROI) levels. To investigate whether the mitogen-activated protein kinase (MAPK) signalling pathway is involved in the downregulation of IL-18 production, cells were pretreated with SB203580, an inhibitor of p38 MAPK, prior to the addition of vitamin C. This pretreatment blocked the decrease in IL-18 production. However, vitamin C treatment enhanced the expression of phosphorylated p38 MAPK. Taken together, we conclude that vitamin C increases intracellular ROI levels, and regulates IL-18 production through the MAPK signalling pathway.

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Blotting, Western; Cell Line, Tumor; Densitometry; Down-Regulation; Enzyme Inhibitors; Flow Cytometry; Imidazoles; Immunoprecipitation; Interleukin-18; MAP Kinase Signaling System; Melanoma; Mice; p38 Mitogen-Activated Protein Kinases; Pyridines; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Skin Neoplasms; Time Factors

The biological activity of the novel vitamin C derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), was evaluated in vitro and in vivo. The percutaneous absorption of AA-2G was determined in five Japanese males. The excretion of ascorbic acid (AA) in the subjects administered AA-2G was sustained for a longer period than in the subjects administered ascorbic acid 2-phosphate (AA-2P), which is a conventional vitamin C derivative. An analysis of the distribution of AA in the skin showed that small black specks assumed to be AA were observed in the epidermis even 3 d after applying AA-2G. The melanin synthesis in B16 melanoma cells was inhibited more by AA-2G than by AA-2P, and AA-2G also prevented more UV-induced damage of human skin keratinocytes and fibroblasts than AA-2P did. From these in vivo and in vitro results, it is supposed that the conversion of AA-2G to AA is sustained for a long time compared with that of AA-2P, and that AA-2G is an effective and available compound having vitamin C activity in human subjects.

Topics: Absorption; Ascorbic Acid; Cells, Cultured; Cosmetics; Epidermis; Humans; Hydroxyl Radical; Keratinocytes; Lipid Peroxidation; Male; Melanins; Melanoma; Skin; Sunburn; Tissue Distribution; Tumor Cells, Cultured; Ultraviolet Rays

Many cell types transport vitamin C solely in its oxidized form, dehydroascorbic acid, through facilitative glucose transporters. These cells accumulate large intracellular concentrations of vitamin C by reducing dehydroascorbic acid to ascorbate, a form that is trapped intracellularly. Certain specialized cells can transport vitamin C in its reduced form, ascorbate, through a sodium-dependent cotransporter. We found that normal human melanocytes and human malignant melanoma cells are able to transport vitamin C using both mechanisms. Melanoma cell lines transported dehydroascorbic acid at a rate that was at least 10 times greater than the rate of transport by melanocytes, whereas both melanoma cells and melanocytes transported ascorbate with similar efficiency. Dehydroascorbic acid transport was inhibited by deoxyglucose and cytochalasin B, indicating the direct participation of facilitative glucose transporters in the transport of oxidized vitamin C. Melanoma cells accumulated intracellular vitamin C concentrations that were up to 100 times greater than the corresponding extracellular dehydroascorbic acid concentrations, whereas intracellular accumulation of vitamin C by melanocytes never exceeded the extracellular level of dehydroascorbic acid. Melanoma cells transported dehydroascorbic acid through at least two different transporters, each with a distinct K(m), a finding that agreed well with the presence of several glucose transporter isoforms in these cells. Only one kinetic component of ascorbate uptake was identified in both melanocytes and melanoma cells, and ascorbate transport was sodium dependent and inhibited by ouabain. Both cell types were able to accumulate intracellular concentrations of vitamin C that were greater than the extracellular ascorbate concentrations. The data indicate that melanoma cells and normal melanocytes transport vitamin C using two different transport systems. The transport of dehydroascorbic acid is mediated by a facilitated mechanism via glucose transporters, whereas transport of ascorbic acid involves a sodium-ascorbate cotransporter. The differential capacity of melanoma cells to transport the oxidized form of vitamin C reflects the increased expression of facilitative transporters associated with the malignant phenotype.

Topics: Ascorbic Acid; Biological Transport, Active; Cells, Cultured; Cytochalasin D; Dehydroascorbic Acid; Deoxyglucose; Dose-Response Relationship, Drug; Humans; Immunohistochemistry; Lithium Chloride; Melanocytes; Melanoma; Models, Biological; Monosaccharide Transport Proteins; Ouabain; Sodium-Potassium-Exchanging ATPase; Sucrose; Time Factors; Tumor Cells, Cultured

In this paper, we show that human neuroectodermal cells exposed to 1-5 mM hydrogen peroxide or 10 nM-1 mM ascorbate die by programmed cell death induced by oxidative stress. The cell death by peroxide occurs within 4 h and involves approximately 80% of B-mel melanoma cells, while ascorbate causes cell death of approximately 86% of B-mel cells within 24 h. SK-N-BE(2) neuroblastoma cells are more resistant, 32% and 43% cell death for peroxide and ascorbate, respectively. In all cases, cell death causes hypodiploic DNA staining, evaluated by flow cytometry. Both cell lines can efficiently metabolise ascorbate due to significant levels of NADH-dependent semidehydroascorbate reductase and glutathione-dependent dehydroascorbate reductase. The cell death observed suggests a pro-oxidant, rather than anti-oxidant, role for ascorbic acid at physiological concentrations under these experimental conditions.

Topics: Apoptosis; Ascorbic Acid; Dehydroascorbic Acid; DNA, Neoplasm; Humans; Hydrogen Peroxide; Melanoma; Neuroblastoma; Oxidative Stress; Oxidoreductases; Tumor Cells, Cultured

The effect of a mixture of vitamins in modifying the efficacy of commonly used drugs in the treatment of human melanoma has not been studied. Vitamin C and d-alpha-tocopheryl succinate (alpha-TS) alone reduced the growth of human melanoma (SK-30) cells in culture, whereas beta-carotene (BC), 13-cis-retinoic acid (RA), or sodium selenite alone was ineffective. RA caused morphological changes, as evidenced by flattening of cells and formation of short cytoplasmic processes. A mixture of four vitamins (vitamin C, BC, alpha-TS, and RA) was more effective in reducing growth of human melanoma cells than a mixture of three vitamins. The growth-inhibitory effect of cis-platin, decarbazine, tamoxifen, and recombinant interferon-alpha 2b was enhanced by vitamin C alone, a mixture of three vitamins (BC, alpha-TS, and RA), and a mixture of four vitamins (vitamin C, BC, alpha-TS, and RA) that contained 50 micrograms/ml of vitamin C. These data show that a mixture of three or four vitamins can enhance the growth-inhibitory effect of currently used chemotherapeutic agents on human melanoma cells.

Topics: Ascorbic Acid; beta Carotene; Carotenoids; Cell Division; Cisplatin; Dacarbazine; Drug Screening Assays, Antitumor; Humans; Interferon alpha-2; Interferon-alpha; Isotretinoin; Melanoma; Recombinant Proteins; Tamoxifen; Tocopherols; Tumor Cells, Cultured; Vitamin E; Vitamins

The levels of tyrosinase mRNA and tyrosinase activity were analyzed in two amelanotic melanoma cell lines, D1(178) (hamster) and G-361 (human). Neither tyrosinase mRNA nor tyrosinase activity were detected in D1(178) cells. On the other hand, both tyrosinase mRNA and weak tyrosinase activity were detected in G-361 cells. Assuming that the different types of melanogenic inhibitors affected melanogenesis in these two amelanotic melanoma cells in different manners, we performed a screening of melanogenic inhibitors in these two cell lines. As an isolated tyrosinase suppressive melanogenic inhibitor, ascorbic acid and glutathione were identified from D1(178) cells and G-361 cells, respectively. Furthermore, lactic acid was identified from D1(178) cells as an isolated tyrosinase non-suppressive melanogenic inhibitor. B-16 mouse melanotic melanoma cells were depigmented by treatment with lactic acid. The melanogenesis suppression by lactic acid in B-16 cells was found to be due to inhibition of tyrosinase gene expression.

Topics: Animals; Ascorbic Acid; Cell Line; Cricetinae; Glutathione; Humans; Lactates; Lactic Acid; Melanins; Melanoma; Monophenol Monooxygenase; RNA, Messenger; Transcription, Genetic

The deuterated benzaldehyde derivative zilascorb(2H), 5,6-O-benzylidene-d-L-ascorbic acid, was administered once daily by i.v. injection in nude mice with grafted tumours of a human malignant melanoma (E.E.) and ovarian carcinoma (OVCAR-3) origins. Like benzaldehyde, zilascorb(2H) has been shown to induce protein synthesis inhibition at otherwise non-toxic doses in cells grown in vitro, and acts reversibly in the sense that protein synthesis returns to normal shortly after removal of the drug. The present data indicate that daily injections with zilascorb(2H) induce a tumour volume growth inhibitory effect in both tumour xenografts studied. Furthermore, from histological examinations of each single tumour it was found that tumours of drug-treated animals, although smaller than those of placebo-treated (i.e. control) animals, had, on average, a higher necrotic fraction than control tumours. Thus, it is concluded that zilascorb(2H) induces tumour necrotisation and not just inhibition of the rate of tumour cell production. Continued measurement of tumour volume after ended treatment with zilascorb(2H) indicated that surviving tumour cells resumed their normal growth rate immediately. The reversibility of the effect induced by this compound, earlier observed in vitro only, is therefore here confirmed to be valid also in two different tumour xenografts in vivo. The present data accords well with the assumption that protein synthesis inhibition is the primary cellular effect of zilascorb(2H) in vivo. We therefore conclude that zilascorb(2H)-induced cancer cell lethality in tumour xenografts probably comes as a secondary consequence of prolonged protein synthesis inhibition.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Benzylidene Compounds; Cell Division; Deuterium; Dose-Response Relationship, Drug; Female; Humans; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Necrosis; Neoplasm Proteins; Neoplasm Transplantation; Ovarian Neoplasms; Transplantation, Heterologous

A phenolic amine compound, 4-S-cysteaminylphenol (4-S-CAP), was found to cause a selective destruction of follicular melanocytes. It was also recently found that 4-S-CAP can be a substrate for both tyrosinase and plasma monoamine oxidase (MAO). Both of these enzymes are capable of producing cytotoxic intermediates through their interaction with 4-S-CAP. To study the mechanism of selective melanocytotoxicity of phenolic amine compounds, we compared the in vivo depigmenting potency of 4-S-CAP and its three analogues; i.e., 4-S-HomoCAP, alpha-methyl(Me)-4-S-CAP and N,N-dimethyl(DiMe)-4-S-CAP, using black hair follicles. All four of these phenolic amine compounds possessed depigmenting potency. In this study we examined the kinetics of tyrosinase and MAO by these four compounds. 4-S-CAP and 4-S-HomoCAP were the substrates of both tyrosinase and MAO, whereas alpha-Me-4-S-CAP and N,N-DiMe-4-S-CAP were the substrates of tyrosinase alone. The rate of o-quinone formation by tyrosinase was not in parallel to the in vivo depigmenting potency of the tested compounds. It is therefore indicated that plasma MAO is not the enzyme directly responsible for the production of the melanocytotoxic intermediates from the phenolic amine compounds. We also found that the observed in vivo depigmentation results from complex processes involving the amount of o-quinone formed and the intracellular interaction of o-quinone with protein species.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Basidiomycota; Cell Line; Cysteamine; Kinetics; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Monoamine Oxidase; Monophenol Monooxygenase; Oxidation-Reduction; Pigmentation; Quinones

Ascorbic acid has been reported to play a role in treatment and prevention of cancer. This study was carried out to determine the effect of ascorbate on growth of normal LLCMK cells and transformed BL6 cells in cell culture and on the growth of BL6 melanomas in vivo. Ascorbic acid levels were also measured to determine the effect of tumor growth and supplementary ascorbate on cellular ascorbic acid levels. Ascorbate addition at levels of up to 200 micrograms/ml was found to inhibit the in vitro growth of BL6 cells but not of LLCMK cells. Ascorbic acid levels in both cell types were very similar. The presence of tumors was found to reduce liver ascorbic acid levels in mice. Supplementary dietary ascorbate increased liver and tumor ascorbic acid levels and also reduced the growth of BL6 melanomas transplanted in C57 mice. Ascorbate thus appears to play a role in suppression of BL6 melanoma growth.

Topics: Animals; Ascorbic Acid; Cell Division; Cell Line, Transformed; Liver; Melanoma; Mice; Neoplasm Transplantation; Tumor Cells, Cultured

Topics: Animals; Ascorbic Acid; Cell Line; Cell Survival; Humans; Melanoma; Microscopy, Electron; NAD; Oxidation-Reduction; Tumor Cells, Cultured

Both ascorbic acid and the 1-series prostaglandins have been reported to be important regulators of cell growth and since ascorbic acid also increases the synthesis of the 1-series prostaglandins, it is possible that the effects of ascorbic acid on cell growth might be mediated by changes in 1-series prostaglandin synthesis induced by ascorbic acid. This study attempted to examine this possible relationship. The effects of ascorbic acid, prostaglandin E1 and the essential fatty acid precursors of the prostaglandins, linoleic acid and gamma-linolenic acid on the in vitro growth of transformed BL6 murine melanoma cells and untransformed monkey kidney (LLCMK) cells was determined. The effects of ascorbic acid addition on the growth inhibitory effect of the essential fatty acids and on the activity of delta-6-desaturase, a key enzyme in 1-series prostaglandin synthesis were also examined. Addition of ascorbic acid, prostaglandin E1 and both essential fatty acids was found to reduce BL6 growth while PGE1 and to a lesser extent the essential fatty acids reduced LLCMK cell growth. The growth inhibitory effect of the essential fatty acids was enhanced by ascorbic acid which was also found to stimulate delta-6-desaturase activity in BL6 cells. The growth inhibitory effect of ascorbic acid on BL6 cells may thus be mediated by changes in prostaglandin synthesis through an association with the metabolism of the essential fatty acid precursors of the prostaglandins.

Topics: Animals; Ascorbic Acid; Cell Division; Fatty Acid Desaturases; Growth Inhibitors; Haplorhini; Linoleoyl-CoA Desaturase; Melanoma; Mice; Prostaglandins; Tumor Cells, Cultured

The single and combined effects of (a) dietary restriction of phenylalanine and tyrosine, (b) levodopa methylester chemotherapy, and (c) megadose sodium ascorbate supplementation on experimental metastasis was determined in B16-BL6 melanoma. Dietary restriction and levodopa methylester therapy inhibited tumor outgrowth, whereas ascorbate alone was inactive. In combination, however, the effect of dietary restriction and levodopa methylester chemotherapy was augmented by sodium ascorbate. Tumor cells surviving this combination therapy (treated population) were isolated from the lungs of treated mice, and proved to be tumorigenic when inoculated SC into the back of naive mice. The resulting tumors grew more slowly than those produced by inoculation of similarly isolated control cells (control population), irrespective of whether the diet was adequate or deficient in phenylalanine and tyrosine. Failure of the treated tumor cell population to exhibit reduced sensitivity to the combination chemotherapy or, unlike the control population, to exhibit variation in pigmentation levels, suggests that the restriction of phenylalanine and tyrosine during drug therapy alters the tumor response to reduce heterogeneity and perhaps interferes with the emergence of drug resistance.

Topics: Animals; Ascorbic Acid; Diet; Female; Levodopa; Lung Neoplasms; Melanoma; Mice; Mice, Inbred Strains; Phenylalanine; Tyrosine

Treatment with the drug combination of levodopa methylester and benserazide, supplemental ascorbate, and dietary deficiencies of tyrosine and phenylalanine more than doubled the median survival time of female (C57BL/6 X DBA/2)F1 mice bearing B16 melanoma tumors. The mechanism for this antitumor effect was not well defined. This study was designed to test the hypothesis that the antitumor activity of levodopa methylester and ascorbate against B16 melanoma is related to the generation of free radicals of oxygen, which peroxidize lipid constituents of cell membranes leading to cell death. As an indication of lipid peroxidation, the individual and combined effects of drug treatment and ascorbate supplementation on host and tumor malondialdehyde levels were examined in mice fed one of three test diets (commercial, purified, or deficient) containing decreasing amounts of tyrosine and phenylalanine. Malondialdehyde levels were increased in the livers of all untreated tumor-bearing mice, which suggests that the tumor alters host antioxidant defenses. Drug treatment and ascorbate supplementation alone and in combination increased hepatic malondialdehyde levels inversely to the amounts of tyrosine and phenylalanine in the diet, and the effects of drug and ascorbate on malondialdehyde levels were additive. Plasma levels remained unchanged by drug treatment, ascorbate supplementation, or tumors in mice fed the commercial or purified diets. Higher levels were observed only in tumor-bearing mice fed the deficient diet and given both drug treatment and ascorbate supplementation. Changes in tumor malondialdehyde levels generally correlated with the effects of the drug and ascorbate on survival time of mice bearing B16 melanoma. Tumors from mice fed the commercial diet accumulated little malondialdehyde, and therapy was relatively ineffective in this dietary group. In mice fed purified or deficient diets, drug treatment and ascorbate supplementation alone increased survival and tumor malondialdehyde levels, but the level of peroxidation in mice receiving the ascorbate supplementation was low compared to its greater antitumor effect on B16 melanoma. Although ascorbate enhanced the peroxidative activity of the drug on B16 melanoma tumors, the effects of the drug and ascorbate on malondialdehyde levels were not additive. Ascorbate enhanced survival of tumor-bearing mice that were fed the deficient diet and that were treated with drug, which indicated that ascorbate supplemen

Topics: Animals; Ascorbic Acid; Diet; Female; Levodopa; Lipid Peroxides; Liver; Malondialdehyde; Melanoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Phenylalanine; Tyrosine

The present study was conducted to clarify the mechanism responsible for enhancement of the anti-melanoma activity of levodopa methylester by supplemental ascorbate in vivo. 5-Hydroxydopa, a known cytotoxic agent and the major metabolite formed from levodopa in the presence of ascorbate and mushroom tyrosinase in vitro, was assessed for its antitumor activity against i.p. and s.c. inoculated B16 melanoma, P388 leukemia, and L1210 leukemia in mice with and without supplemental ascorbate. Treatment with 5-hydroxydopa failed to significantly increase survival of mice bearing i.p. or s.c. pigmented and non-pigmented B16 melanomas even though it inhibited local tumor growth. Treatment increased survival of both P388 and L1210 leukemias, and this increase was more pronounced in mice bearing i.p. tumors than in mice bearing s.c. tumors. This treatment significantly decreased final tumor weight of both leukemias implanted s.c., and inhibited ascites formation in mice inoculated with i.p. tumors. Ascorbate supplementation decreased or abrogated the effect of 5-hydroxydopa on survival in mice bearing i.p. or s.c. leukemia tumors and decreased survival relative to control mice bearing i.p. or s.c. pigmented and s.c. non-pigmented tumors. It did not affect survival of treated mice bearing i.p. non-pigmented melanoma tumors. Ascorbate supplementation did not modify the effect of 5-hydroxydopa treatment on primary s.c. tumor growth in mice bearing melanoma or leukemia tumors nor did it affect ascites formation in treated mice bearing i.p. leukemia tumors. The lack of correlation between the observed inhibition of primary tumor growth and the absence of an effect on survival in 5-hydroxydopa treated mice bearing i.p. melanoma may relate to an inability of this drug to interfere with tumor metastasis. These data argue against a role for 5-hydroxydopa as a metabolically derived cytotoxic formed in situ during concurrent treatment with levodopa methylester and supplemental ascorbate.

Topics: Animals; Ascorbic Acid; Dihydroxyphenylalanine; Drug Interactions; Female; Leukemia L1210; Leukemia P388; Male; Melanoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA

Marked sensitivity of tumor tolerance to blood glucose level is demonstrated in a mouse model of human breast cancer. A theory is cited that explains the high association of hyperglycemia with malignancy, especially breast cancer, via glycemic modulation of cellular immunity. Three groups of BALB/C mice were injected with an aggressive mammary tumor and placed on three dietary regimens designed to produce three different glycemic levels. Mortalities 70 days after injection were 16 of 24 hyperglycemic mice, 8 of 24 normoglycemic, and 1 of 20 hypoglycemic (chi-square p less than .005). Taken together with other experiments and human data discussed briefly, this result suggests that glycemic modulation of tumor tolerance should be evaluated in human trials.

Topics: Animals; Ascorbic Acid; Blood Glucose; Disease Models, Animal; Female; Leukocytes; Mammary Neoplasms, Experimental; Melanoma; Mice; Mice, Inbred BALB C; Phagocytosis

The medium of cultured melanoma cells was studied for tyrosine hydroxylation and dopa-oxidizing activity. The supernatant obtained after centrifugation at 100 000 g for 2 hours was treated with ammonium sulphate, and the precipitate obtained between 35 and 50% saturation was used. Dopa was determined as the product of tyrosine hydroxylation and 5-S-cysteinyldopa as the product of dopa oxidase activity. Determinations were performed with HPLC and electrochemical detection. Our preparation of culture medium of cells showed the following. 1) No hydroxylation of tyrosine in the absence of co-factor. 2) Hydroxylation of L-tyrosine in the presence of dopamine. No hydroxylation with boiled medium. Minimal effect of catalase on hydroxylation. 3) Hydroxylation of tyrosine in the presence of ascorbic acid. Hydroxylation was catalyzed also with boiled medium. Catalase strikingly diminished hydroxylation. 4) Oxidation of L-dopa to dopaquinone determined as its main reaction product with cysteine, 5-S-cysteinyl-dopa. There was negligible oxidation with boiled medium. 5) With dopamine as co-factor the catalysis of tyrosine hydroxylation was stereospecific for L-tyrosine. Dopa oxidase activity was also stereospecific for L-dopa.

Topics: Ascorbic Acid; Catechol Oxidase; Cells, Cultured; Chromatography, High Pressure Liquid; Culture Media; Dihydroxyphenylalanine; Humans; Hydroxylation; Melanoma; Monophenol Monooxygenase; Oxidation-Reduction; Stereoisomerism; Tyrosine

Ascorbate-Cu2+ shows considerable cytotoxicity for human melanoma cells at a dose which has very little effect on human fibroblasts. Ascorbate itself inhibits DNA synthesis in melanoma cells but does not fragment the parental DNA. However, the combined action of ascorbate-Cu2+ generates fragmentation of the parental DNA due to the induction of alkali-labile bonds in the DNA. In contrast, if DNA polymerase alpha is inhibited by aphidicolin prior to treatment with ascorbate-Cu2+ one cannot detect the fragmentation of the DNA. The generated fragments show a discrete appearance in agarose gel electrophoresis with a single-stranded size of approximately 5 kb. When fibroblasts were analyzed using the same experimental protocol it was not possible to detect the fragmentation of the DNA.

Topics: Aphidicolin; Ascorbic Acid; Cells, Cultured; Copper; Diterpenes; DNA Polymerase II; DNA Replication; DNA, Neoplasm; Electrophoresis, Agar Gel; Fibroblasts; Humans; Melanoma

We report here the single and combined antitumor activity on B16 melanoma in female C57BL/6 X DBA/2F1 mice bearing s.c. tumors of sodium ascorbate, carbidopa-levodopa methyl ester, and dietary phenylalanine and tyrosine deficiency. Groups of 15 mice were fed continuously one of three test diets both with and without sodium ascorbate (30 mg/ml) in the drinking water beginning 2 weeks before inoculation of 10(6) melanoma cells. The test diets included the following amounts of tyrosine-phenylalanine: commercial, 1.09 and 0.64%; purified, 0.6 and 0.3%; and deficient, 0.08 and 0.04%. Drug-treated groups received daily injections of carbidopa (100 mg/kg) and levodopa methyl ester (1000 mg/kg) i.p. for 15 days beginning 1 day after tumor transplant. Tumor growth curves and median survival time were determined. Ascorbate stimulated tumor growth in the commercial diet group. In mice fed the purified diet, ascorbate inhibited growth in some tumors, while it had no effect on others. Ascorbate inhibited tumor growth in mice fed the deficient diet, which itself severely inhibited tumor growth, and in this group increased survival by 82%. Drug treatment had little effect on tumor growth and survival of mice fed the commercial diet, but it significantly decreased growth and moderately increased survival of mice fed the purified diet. The deficient diet enhanced drug activity and increased survival of tumor-bearing mice by 73%. Combined therapy had little effect in mice fed the commercial diet;l however, mice fed the purified diet and receiving drug and ascorbate had smaller tumors and lived 55% longer. In mice fed the deficient diet, the combination retarded tumor growth and increased survival dramatically by 123%. These data indicate that adding ascorbate and restricting tyrosine and phenylalanine in combination with levodopa methyl ester therapy may become an important strategy for treating malignant melanoma.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Carbidopa; Cell Line; Diet; Drug Synergism; Drug Therapy, Combination; Female; Injections, Intraperitoneal; Levodopa; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms, Experimental; Phenylalanine; Tyrosine

Hemicalcium ascorbate (Ca-Asc, 51 mM, 1% wt/vol), added to the drinking water, had the following effects in DBA/2 mice inoculated with 10(5) S91 (Cloudman) melanoma cells: 1) it delayed the appearance of visible tumors by 2-4 weeks; 2) it increased the survival rate at three months after tumor challenge by 12-50%; 3) it had no significant effect on the rate of tumor growth once the size of the tumors had reached 10 mm3; 4) the inhibition was maximal when the treatment with Ca-Asc was started at least one week prior to the inoculation of cells 5) when free ascorbic acid was used instead of Ca-Asc, the animals consumed 50% less water, they became dehydrated and the treatment was less effective; 6) Ca++ (51 mM) alone had no significant inhibitory effect.--Since Ca Asc (1 mM) was not toxic to S91 melanoma cells in vitro, we suggest that prophylactic treatment of the animals with Ca-Asc inhibited tumor development by increasing the resistance of the host.

Topics: Animals; Ascorbic Acid; Calcium; Cell Division; Copper; Female; Melanoma; Mice; Mice, Inbred DBA; Neoplasm Transplantation; Neoplasms, Experimental; Time Factors

Six of eight human melanoma lines showed increased sensitivity to killing by dopa and by ascorbate:copper compared with two fibroblast strains and four other human cell lines of nonmelanoma origin. Catechol, epinephrine, and alpha-methyldopa, but not 5,6-dihydroxyindole, exhibited a similar degree of selectivity. Toxicity was greatly reduced when brief exposure times or high cell densities were used. Depending upon culture conditions, melanoma cells accumulated more [3H]dopa- and [14C]ascorbate-derived isotopic label within the first five min than fibroblasts, but after one hr this difference was less marked. The catalase activity in melanoma cells was not less than that in fibroblasts. Using two independent methods to determine each type of damage, dopa and ascorbate:copper were found to induce DNA breaks in both cell types but not DNA repair synthesis or DNA interstrand cross-links. More DNA breaks were found in melanoma cells (two lines) than in fibroblasts. Semiconservative DNA synthesis was inhibited immediately, recovered within six hr, and in melanoma cells, was again inhibited after 24 hr. RNA synthesis was inhibited less than DNA synthesis. Human cell lines with differential sensitivity to gamma-radiation, ultraviolet light, cross-linking agents, or monofunctional alkylating agents, exhibited normal survival levels when treated with dopa or ascorbate:copper.

Topics: Ascorbic Acid; Catalase; Cell Line; Cell Survival; Copper; Dihydroxyphenylalanine; DNA Repair; DNA, Neoplasm; Fibroblasts; Humans; Melanoma; RNA, Neoplasm

Vitamin C has been suggested and disputed as an anti-cancer agent. For cells in culture, no preferential effect against any type of cancer has yet been demonstrated. Our aim here is to show that vitamin C is selectively toxic to at least one type of malignant cell--a melanoma--at concentrations that might be attained in humans. Copper ions react with ascorbate and generate free radicals in solution. Ascorbate when combined with copper rapidly reduces the viscosity of DNA solutions and has exhibited some carcinostatic effects on transplanted sarcoma 180 tumours in mice. We reasoned that the elevated copper concentration in melanoma could result in a more selective toxicity for ascorbate.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Cell Division; Cell Line; Copper; Cricetinae; Dose-Response Relationship, Drug; Drug Synergism; Humans; Hydrogen-Ion Concentration; Melanoma; Mice; Neoplasms, Experimental

Topics: Animals; Ascorbic Acid; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred C57BL; Neoplasms, Experimental

Reducing agents had no effect on the oxidation of 3,4-dihydroxyphenylalanine (DOPA) to quinone by Mycobacterium leprae; no quinone formation by o-diphenoloxidase of mammalian or plant origin was detected under similar experimental conditions. Ascorbic acid and reduced glutathione prevented further oxidation and polymerization of the quinone to melanin by M. leprae; cysteine was less effective. In the presence of reducing agents, the quinone (indole-5,6-quinone) formed from DOPA by M. leprae was not reduced back to diphenol. On the other hand, the quinone (dopachrome) produced from DOPA by mammalian or plant phenolase was rapidly decolorized by reducing agents. Oxidized glutathione and cystine had little effect on o-diphenoloxidase from all of the three sources. Cyanide, which completely inhibited mammalian and plant phenolases, had only a partial effect on the enzyme in the bacilli. Various lines of evidence suggest that the properties of o-diphenoloxidase in M. leprae are different from those of similar enzymes obtained from other sources.

Topics: Animals; Ascorbic Acid; Azides; Basidiomycota; Catechol Oxidase; Cell-Free System; Cyanides; Cysteine; Cystine; Dihydroxyphenylalanine; Glutathione; Humans; Manometry; Melanins; Melanoma; Mice; Mycobacterium leprae; Oxidation-Reduction; Quinones; Skin; Species Specificity; Spectrophotometry; Spleen; Stereoisomerism; Tissue Extracts; Tyrosine

Topics: Animals; Animals, Newborn; Ascorbic Acid; Colorimetry; Culture Techniques; Electron Spin Resonance Spectroscopy; Heart; Melanoma; Mice; Sucrose

Topics: Animals; Ascorbic Acid; Catechol Oxidase; Dihydroxyphenylalanine; Hot Temperature; Melanoma; Mice; Neoplasm Transplantation; Tritium; Tyrosine; Ultracentrifugation

Topics: Adrenocorticotropic Hormone; Animals; Ascorbic Acid; Chromatophores; Colchicine; Copper; Culture Techniques; Dihydroxyphenylalanine; Hydroquinones; Melanins; Melanocyte-Stimulating Hormones; Melanoma; Melatonin; Mice; Motion Pictures; Pigmentation; Tyrosine

Topics: 5-Hydroxytryptophan; Animals; Ascorbic Acid; Carbamates; Cricetinae; Cyanides; Dihydroxyphenylalanine; Enzymes; In Vitro Techniques; Melanoma; Mixed Function Oxygenases; Tritium; Tyrosine; Tyrosine Decarboxylase

Topics: Animals; Ascorbic Acid; Dehydroascorbic Acid; Humans; Melanoma; Mice; Neoplasms